Pertussis toxin ptx

Monoclonal antibody (mAb) anti‐His tag (clone AD1.1.10, FITC‐labelled) was purchased from LS Biosciences, and anti‐CD62L (clone Dreg‐56, FITC‐labelled) and anti‐CD11b (clone ICRF44, APC‐labelled) were purchased from BD. The peptide MMK‐1 (H‐LESIFRSLLFRVM‐OH) was synthesised by Sigma, and WKYMVM was synthesised by Bachem AG (Switzerland). WRWWWW‐NH2 (WRW4) and Pertussis toxin (PTX) were purchased from Tocris. Formyl‐methionyl‐leucyl phenylalanine (fMLP), tumour necrosis factor α, and cytochalasin B were from Sigma‐Aldrich. Fluo‐3‐AM (acetoxymethyl ester) and Calcein‐AM were purchased from Thermo Fisher.

pBABE-puro-NLS-LSSmKate2 was a gift from Vladislav Verkhusha (Addgene plasmid #34586). Gα_i1 (#GNAI100000), Gα_i2 (#GNAI200000), Gα_i3 (#GNAI300000), Gα_OA (#GNA0OA0000), Gα_OB (#GNA0OB0000), Gα_Ss (#GNA0SS0000), Gα_SL (#GNA0SL0000), Gα₁₂ (#GNA1200000), Gα₁₃ (#GNA1300001), Gα_q (#GNA0Q00000), Gα₁₅ (#GNA1500000) Gγ1 (#GNG0100000), Gγ2 (#GNG0200000), Gγ9 (#GNG0900000), Gβ1 (#GNB0100000), β2-adrenergic receptor (#AR0B200000), Thromboxane A2 receptor (#TXA2R00000) and Histamine 3 receptor (#HRH0300000) were purchased from cDNA Resource Centre and are in the pcDNA3.1(+) backbone. The pNLF1-N vector (#N1351) and pGlosensor-22F cAMP plasmid (#E2301) were purchased from Promega. Pertussis toxin (PTX; #3097) was purchased from Tocris, Bristol, UK, Cholera Toxin (CTX; #C8052) from Merck, Rahway, NJ, USA and YM-254890 (#257-00631) from FUIJIFILM Wako Chemicals, Osaka, Japan. Histamine dihydrochloride (Histamine; #3545) was purchased from Bio-Techne, Minneapolis, MN, USA; Isoprenaline hydrochloride (Isoprenaline; #I5627), 9,11-Dideoxy-11α,9α-epoxymethanoprostaglandin F2α (U46619; #D8174), 3-isobutyl-1-methylxanthine (IBMX; #I7018), Forskolin (FSK; #F6886) and Poly-D-Lysine (PDL; #2780) from MERCK. Nano-Glo^® Vivazine™ substrate (#N2581) and NanoBRET™ Nano-Glo^® Substrate (#N1571) were purchased from Promega, Madison, WI, USA.

HEK293 cells obtained from ATCC were used to generate HEK293-human GPR142 and HEK293-mouse Gpr142 stable cell lines. Briefly, HEK293 cells were transfected with pcDNA3.1 plasmids containing hGPR142 (RefSeq XM_005257305.2) or mGpr142 (RefSeq XP_006533129) insert using Lipofectamine Reagent (Invitrogen), and clones were selected with Dulbecco’s modified Eagle’s medium (DMEM) (Sigma) supplemented with 10% fetal bovine serum (FBS), 1% Antibiotic-Antimycotic, and 800 μg/ml G418 (Gibco). Stable cell lines were maintained in DMEM supplemented with 10% FBS, 1% Antibiotic-Antimycotic, and 800 μg/ml G418 and grown at 37°C in a humidified atmosphere of 5% CO₂/95% air. Pertussis Toxin (PTX, Tocris Bioscience) was used to pre-treat cells overnight at a concentration of 100 ng/ml to inhibit Gi signaling. UBO-QIC was ordered from Institute of Pharmaceutical Biology, University of Bonn, and used to pre-treat cells for 1 hour at a concentration of 0.3 μM to inhibit Gq/11 signaling. PTX and UBO-QIC at the tested concentrations did not affect cell viability as measured by LDH release or cellular ATP content.

DAMGO, U50488H, SNC80, CGS21680, AVP (arginine vasopressin), IBMX, Forskolin, stromal cell-derived factor 1 (SDF-1 or CXCL12), carbamoylcholine chloride (carbachol), quinpirole hydrochloride, SKF 81297, ionomycin calcium salt, were purchased from Tocris Bioscience (Bristol, UK) and diluted in DMSO (diméthylsulfoxyde) at 10⁻² M (except SDF1 at 125 µM and IBMX at 200 mM) for frozen stock aliquots and coelenterazine H substrate from Interchim (Montluçon, France) was diluted in 100% ethanol and kept at −20°C. Protease/Phosphatase Inhibitor Cocktail were purchased from Cell Signaling Technology (Leiden, Netherlands). Pertussis toxin (PTX), purchased from Tocris, were diluted in water at 0.1 µg/µl and stored at 4°C. Morphine HCl was purchased from Francopia (Paris, France). Phenylmethanesulfonyl fluoride (PMSF) was diluted in isopropyl alcohol at 200 mM and stored at −20°C. Compound 19 was generously synthetized by Domain Therapeutics (Illkirch, France). For in vitro studies, it was diluted in DMSO (diméthylsulfoxyde) at 10⁻² M and frozen at −20°C. For in vivo studies, Compound 19 was kept as a powder at 4°C and diluted in a saline solution before ICV administration (NaCl 9%).

Compound C was purchased from Calbiochem (San Diego, CA, United States). Wortmannin was purchased from Selleckchem (Houston, TX, United States). Gö6983 was purchased from EMD Millipore (Billerica, MA). Fluo-4 AM was purchased from Invitrogen (Camarillo, CA, United States). Pertussis Toxin (PTX) and Gallein were purchased from Tocris Bioscience (Bristol, United Kingdom). Ryanodine was purchased from Cayman Chemical (Ann Arbor, MI, United States). 1,2-Bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM, Caffeine, a chelator of Ca²⁺), U73122 and 2-APB were purchased from Sigma (St. Louis, MO, United States). GLUT4 antibody (#2213), Akt antibody (#9272), phospho-Akt (Ser473) (193H12) antibody (#4058) and phospho-PKC (pan) (Thr410) antibody (#2060) were purchased from Cell Signaling Technology (Beverly, MA, United States). The Anti-c-myc mouse monoclonal antibody (#M10117) and FITC antibody (#M10422) were both purchased from TransGen Biotech (Beijing, China).

Naïve C57BL/6 mice or MKP-2^−/− and MKP-2^+/+ littermates were immunized subcutaneously on day 0 with 100 μl of 100 μg MOG_35–55 (ChinaPeptides Co Ltd) emulsified in complete freunds adjuvant (CFA; Sigma) supplemented with 3.65 mg Mycobacterium tuberculosis (BD Biosciences). In addition, each mouse received 100 ng pertussis toxin (PTX; Tocris Bioscience) in 100 μl PBS injected intraperitoneally on day 0 and again on day 2. Mice were monitored daily for signs of disease development and given a clinical score based on the following evaluation system: 0 = no clinical sign; 0.5 = partial loss of tail tone; 1.0 = complete loss of tail tone; 1.5 = altered gait; 2.0 = hind limb weakness; 2.5 = paralysis of one leg; 3.0 = hind limb paralysis; 3.5 = hind limb paralysis with significantly reduced mobility; 4.0 = forelimb involvement; 5.0 = moribund.